Absence of Toxoplasma gondii in 100% Iberian products from experimentally infected pigs cured following a specific traditional process.

Infection with Toxoplasma gondii in humans has usually been related to the consumption of raw, undercooked or cured meat. Our study is based on the detection of T. gondii in cured legs and shoulders made from 100% Iberian sows fed mainly with acorn and raised as outdoor livestock in Aracena (Spain), which having been elaborated following a specific curing process (time period and location). An outdoor farm with a total of 636 animals was studied, showing a seroprevalence of 10% for the parasite T. gondii. Twenty individuals were chosen to be experimentally infected and slaughtered 60 days post-infection. Their legs and shoulders were processed to make 100% Iberian ham legs and shoulders. The meat ready to be eaten was analyzed by quantification and viability assays using magnetic capture real-time qPCR and bioassay techniques proving that this specific traditional "Cinco Jotas" curing process 100% Iberian ham is strong enough to eliminate the parasite T. gondii, resulting in a safe product for consumers.

Clinical and epidemiological descriptions from trichinellosis outbreaks in Bulgaria.

Bulgaria is one of European countries where trichinellosis continues to be regularly diagnosed and registered. The clinical and epidemiological features of 72 cases of trichinellosis associated with five outbreaks caused by Trichinella spiralis and Trichinella britovi between 2009 and 2011, are described. At hospital admission, patients were often initially treated with antibiotics, without any improvement. A range of signs and symptoms were recorded, including: myalgia, elevated temperature, arthralgia, difficulty with movement, facial oedema, conjunctival hyperaemia, ocular haemorrhages, diarrhoea, skin rash, headache, and fatigue. Due to the variable clinical course of the disease, the diagnostic process for trichinellosis is often complex and difficult. This means the diagnosis may be established late for an appropriate treatment, potentially leading to a severe course of the disease with complications. Laboratory abnormalities were expressed by marked eosinophilia (97.2%), leucocytosis (70.8%), elevated serum creatine phosphokinase levels (82%), and antibody-positive results by ELISA and indirect hemagglutination. Patients were treated with albendazole (Zentel) 10 mg/kg for 7-10 days. In two outbreaks, the aetiological agent was T. spiralis, in one outbreak T. britovi, and an unknown Trichinella species in the fourth outbreak. The sources of infection were domestic pigs, probably fed with scraps and offal of wild game. In one outbreak, T. spiralis was also detected in brown rats trapped close to where the pig had been raised in the backyard. These epidemiological factors are relevant in considering implementation of targeted control programmes.

Detection and Molecular Characteristics of Toxoplasma gondii DNA in Retail Raw Meat Products in Poland.

Raw and undercooked meat are regarded as important sources of Toxoplasma gondii infection of people in Europe; however, data concerning this issue in Poland are still insufficient. The aim of this study was to determine the prevalence of T. gondii DNA isolated from raw meat products retailed in Poland. The molecular characteristics of detected DNA were also performed. Samples of cured bacon, raw or smoked sausages, ham, and minced meat were examined for the presence of T. gondii DNA. Samples were digested by pepsin solution, followed by the DNA isolation. Nested and real-time polymerase chain reaction (PCR) was performed based on the amplification of 35-fold-repetitive B1 fragment gene of T. gondii. For selected B1-positive samples, multiplex PCR was performed using SAG1, SAG2 (5'-SAG2 and 3'-SAG2), altSAG2, SAG3, GRA6, BTUB, C29-2, and L358 genetic markers. Amplicons were sequenced and analyzed with NCBI database. Among 3223 examined samples, 175 (5.4%) were PCR positive. The highest percentages of positive results were found for samples originating from south-east regions of Poland-Podkarpackie (17.9%), Malopolskie (12.6%), and Lubelskie (10.8%) (p < 0.001). The percentages of positive results for particular types of meat products-sausages, smoked meat products, ham, and minced meat-ranged from 4.5% to 5.8% and the differences between them were not significant (p > 0.05). Sequence analysis of selected B1-positive samples demonstrated mostly the alleles of clonal type III (49.0%), and less-type II (17.3%), and type I (10.2%) based on nine used genetic markers. The combinations of types I/II or II/III or I/III alleles at different loci were also found in 23.5% of cases. Detection of T. gondii DNA in raw meat products may indicate the potential health threat for consumers in Poland; however, for complete risk assessment of T. gondii infection, the additional studies, including detection of live parasite, are needed.

Risk Assessment of Human Toxoplasmosis Associated with the Consumption of Pork Meat in Italy.

Toxoplasmosis is a cosmopolitan disease and has a broad range of hosts, including humans and several wild and domestic animals. The human infection is mostly acquired through the consumption of contaminated food and pork meat has been recognized as one of the major sources of transmission. There are, however, certain fundamental differences between countries; therefore, the present study specifically aims to evaluate the exposure of the Italian population to Toxoplasma gondii through the ingestion of several types of pork meat products habitually consumed in Italy and to estimate the annual number of human infections within two subgroups of the population. A quantitative risk assessment model was built for this reason and was enriched with new elements in comparison to other similar risk assessments in order to enhance its accuracy. Sensitivity analysis and two alternative scenarios were implemented to identify the factors that have the highest impact on risk and to simulate different plausible conditions, respectively. The estimated overall average number of new infections per year among adults is 12,513 and 92 for pregnant women. The baseline model showed that almost all these infections are associated with the consumption of fresh meat cuts and preparations (mean risk of infection varied between 4.5 × 10-5 and 5.5 × 10-5 ) and only a small percentage is due to fermented sausages/salami. On the contrary, salt-cured meat products seem to pose minor risk but further investigations are needed to clarify still unclear aspects. Among all the considered variables, cooking temperature and bradyzoites' concentration in muscle impacted most the risk.

Prevalence of Toxoplasma gondii DNA in Processed Pork Meat.

Toxoplasma gondii infection may be attributed to the ingestion of pork meat and contaminated water. In southern Brazil, the prevalence of blindness caused by T. gondii is the highest in the world. Our purpose is to determine the frequency of T. gondii DNA in commercial fresh sausage and cured salami samples from Rio Grande do Sul state, south of Brazil. A total of 118 samples (sausage and salami) from 8 different producers were collected and DNA was extracted. Real-time polymerase chain reaction (qPCR) technique was performed to detect T. gondii DNA using B1 marker. The frequency of T. gondii DNA among the total number of samples (sausage and salami) was 39% (46/118). Among these, a higher frequency of positivity was observed in the sausage samples (47.5%) when compared with the salami samples (17%). However, the mean parasite concentration was significantly higher in the salami samples. The prevalence of T. gondii DNA in fresh sausage and cured salami may indicate that infected pigs may be an important source of infections and a public health hazard to be considered.

Lack of viable parasites in cured 'Parma Ham' (PDO), following experimental Toxoplasma gondii infection of pigs.

Twelve Large White pigs were experimentally infected with 1000 Toxoplasma gondii oocysts/each. Serology was carried out at different time points post infection (p.i.) and animals were slaughtered at four months p.i. One of two thighs was examined for T. gondii infection status by PCR and bioassay in mice. The other thigh was processed for Parma ham production. Four thighs were examined after twelve months of curing, four after fourteen months and four were examined after sixteen months. Cured hams were analyzed by PCR, bioassay and in-vitro cultivation on Vero cells followed by real-time PCR. Pigs seroconverted from day 21 p.i. Bioassays were positive for all fresh thighs, but negative for cured hams. PCR was positive for parasite DNA from most thighs both at slaughter and post curing, but parasite growth was not observed following in vitro cultivation and real-time PCR. Results indicate that the curing process of Parma Ham (PDO), when carried out according to the Parma Ham consortium regulations, can inactivate T. gondii tissue cysts. Results would suggest that food-borne transmission of T. gondii to consumers from Parma ham can be excluded.

Meat sources of infection for outbreaks of human trichinellosis.

Trichinellosis is one of the most important foodborne zoonotic diseases, with worldwide distribution. While human risk for trichinellosis has historically been linked to pork, modern pork production systems and slaughter inspection programs have reduced or eliminated pork as a source for trichinellosis in many countries. While pork may no longer pose a significant risk for trichinellosis, many other animal species may be hosts for Trichinella species nematodes and when human consume meat from these animal species, there may be risk for acquiring trichinellosis. This review article describes the various non-pork meat sources of human trichinellosis outbreaks, where these outbreaks have occurred and some of the factors that contribute to human risk. The literature reviewed here provides evidence of the persistence of Trichinella as a human health risk for people who eat meat from feral and wild carnivores and scavengers, as well as some herbivores that have been shown to harbor Trichinella larvae. It points to the importance of education of hunters and consumers of these meats and meat products.

Toxoplasma gondii in raw and dry-cured ham: The influence of the curing process.

The aim of this work was to analyze Toxoplasma gondii in raw hams by mouse bioassay and to evaluate the effect of curing on the viability of the parasite to assess the risk of infection from eating dry-cured ham. After a serology study of 1200 pigs in Aragón (Spain), forty-one naturally infected pigs with different serological titers against T. gondii were selected. Two cured periods (9 and 12 months) were evaluated as well as the influence of the physicochemical composition of hams on T. gondii survival. Although the parasite burden was low, a high number of seropositive pigs with Toxoplasma tissues cysts in raw hams were found (31.6%). Viability of T. gondii was influenced by the curing, with statistically significant differences between fresh and cured hams (p < 0.001). The viability was higher in hams cured for 9 months compared to those cured for 12 months. However, this period of curing resulted in the reduction but not in a complete elimination of the risk. Thus, from a public health point of view, under the conditions of this study it is safer to consume dry-cured ham with periods of curing higher than 12 months. Analysis of physicochemical results did not identify any variable with significant influence on the presence and viability of T. gondii in cured ham, but loss of viability of T. gondii was observed in hams with a lower fat content. Further research is required to validate combinations of salts concentration and time of curing that can be used as preventive measures in the HACCP system of dry-cured ham industry.

Quantification and viability assays of Toxoplasma gondii in commercial "Serrano" ham samples using magnetic capture real-time qPCR and bioassay techniques.

"Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. To make Serrano ham, pork undergoes a salting and a subsequent fermentation process known as curing. Certain pigs used for meat production are an important source of Toxoplasma gondii infection in humans. We have developed a method for quantifying and assaying the viability of the T. gondii present in commercial Serrano ham samples. A magnetic capture method for the isolation of T. gondii DNA and a qRT-PCR were used to estimate the T. gondii burden in 475 commercial samples of "Serrano" ham in two presentation formats: ham pieces and sliced ham. The infectivity capacity of T. gondii in positive samples was assayed in mice. The global prevalence of T. gondii was 8.84%, ranging from 32.35% in one of the companies to 0% prevalence in three other companies. The infectivity assays revealed that only 4.84% of the positive samples were infective. To the best of our knowledge this is the first report focussing on the prevalence of T. gondii in commercial "Serrano" ham. The method described here could be useful for producers to guarantee the safety of their products.

Toxocara optic neuropathy after ingestion of raw meat products.

PURPOSE: To report Toxocara optic neuropathy in five men with histories of ingestion of raw meat, liver, or blood. CASE REPORT: Five men in their 30s to 60s presented with unilateral visual disturbance. All five had a history of ingesting raw meat, liver, or blood products. All five men presented with optic disc edema and three also showed granulomatous infiltration of the retina. With Goldmann or Humphrey visual field testing, two patients showed central scotomas and two exhibited arcuate defects. Serum eosinophil count and total IgE were elevated in four patients. Serum enzyme-linked immunosorbent assay testing was positive for Toxocara canis IgG in all five cases. CONCLUSIONS: This report shows the variable clinical presentations of Toxocara optic neuropathy and the importance of questioning for a history of eating raw meat and blood products as a risk factor.

Survival of experimentally induced Toxoplasma gondii tissue cysts in vacuum packed goat meat and dry fermented goat meat sausages.

Ingestion of raw or undercooked meat is a potential source of human toxoplasmosis. The aim of this study was to determine the viability of Toxoplasma gondii cysts in vacuum packed (VP) goat meat and in dry fermented sausages (DFS), and evaluate certain physical and chemical parameters, like water activity (aw), pH value, content of salt, dry matter and fat. A portion of muscle tissue from experimentally infected animals was used for production of VP meat with or without addition of 2.5% curing salt, and stored at 4 °C or at -20 °C. Results of bioassay showed that, samples of vacuum packed Toxoplasma positive meat without salt addition were alive after six weeks at 4 °C. Incubation at -20 °C supported the viability after 3 h, but not after 4 h. After 7 days in 2.5% of curing salt, samples of T. gondii VP goat meat were still viable, but not after 14 days at 4 °C. All the DFS samples were not positive for infective cysts which mean that, they do not pose a risk of T. gondii transmission. These data suggest that vacuum packaging increases the survival of T. gondii cysts.

[Possible sources of Trichinella infection in the indigenous population of Chukotka].

Statistical methods confirmed that the dietary intake of traditionally made meat from marine mammals and polar bear could cause Trichinella infection in the residents of the communities of the Chukotka Peninsula.

Detection of Toxoplasma gondii in artisanal salted meat products sold in street markets of the Ilhéus-Itabuna microregion.

This study aimed to detect Toxoplasma gondii in artisanal salted meat products sold in street markets in the Ilhéus-Itabuna microregion and to assess the salt concentration used in their preparation and its influence on the parasite's viability. A total of 125 samples of various artisanal meat products sold in street markets located in the Ilhéus-Itabuna microregion were collected during 2021. Serological analysis using indirect hemagglutination (HAI) and molecular analysis (PCR) were performed on these samples to detect the presence of the parasite. Möhr's method was utilized to determine the sodium chloride concentration in the samples. Of all samples, 21 were subjected to a bioassay in albino mice to verify the viability of possible tissue cysts. Among the 125 meat products, 10 (8%) tested positive in the serological analysis including four cured pork sausages, five beef sun-dried meats, and one mixed fresh sausage (pork and chicken). None of 125 samples tested positive in the molecular analysis. On bioassay, all mice tested negative for the presence of the parasite. The NaCl concentration in the positive samples ranged from 2.9% to 8%. The results demonstrated that the salt concentration in the collected samples was sufficient to inactivate the parasite T. gondii.

In vitro assay to determine inactivation of Toxoplasma gondii in meat samples.

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections. However, most non-heated meat products contain salt and additives, which affect T. gondii viability. It was our aim to develop an in vitro method to substitute the mouse bioassay for determining the effect of salting on T. gondii viability. Two sheep were experimentally infected by oral inoculation with 6.5 × 104 oocysts. Grinded meat samples of 50 g were prepared from heart, diaphragm, and four meat cuts. Also, pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution, and digests were inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for up to four weeks and parasite growth was monitored by assessing Cq-values using the T. gondii qPCR on cell culture supernatant in intervals of one week and ΔCq-values determined. Additionally, 500 µL of each digest from the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFNγ KO mice. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. From all untreated meat samples positive ΔCq-values were obtained in the in vitro assay, indicating presence and multiplication of viable parasites. This was in line with the mouse bioassay, with the exception of a negative mouse bioassay on one heart sample. Samples supplemented with 0.6 %-1.2 % NaCl showed positive ΔCq-values over time. The frozen sample and the sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values, which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less.

Trichinellosis in Poland in 2011.

PURPOSE OF THE STUDY: The purpose of the study was assessment of the epidemiological situation of trichinellosis in Poland in 2011. MATERIALS AND METHODS: The evaluation was carried out on the basis of the analysis of data from the bulletin "Infectious diseases and poisonings in Poland. 2010". information from the questionnaires of individual cases and epidemiological investigations of the outbreaks of trichinellosis sent by the Sanitary-Epidemiological Stations and on data from Demographic Research Department of the Central Statistical Office. Cases were classified according to the current case definition. RESULTS: In 2011. Poland recorded 23 cases of trichinellosis cases. The incidence was 0.06/100,000. There were reported 10 confirmed cases and 13 probable. There were no deaths from trichinellosis, but over 91% of infected persons were hospitalized. In 2011 three outbreaks of trichinellosis were recorded. One of them occurred in Dolnoslaskie district, where cases of the infection are recorded sporadically. The epidemiological investigations established that the cause of the outbreak was consumption of wild boar meat products, mainly raw sausage. Trichinella containing meat products are prepared primarily for personal use. but are also distributed among family and friends. CONCLUSION: The annually repeating outbreaks of trichinellosis mainly among family members indicates insufficient awareness of the risk of parasitic diseases caused by consumption of products containing raw or half raw meat of animals. Of the particular importance is to inform those who will give the carcases of animals to test about the limitations of the test method used.

[Trichinellosis in Poland in 2010]. / Wlosnica w Polsce w 2010 roku.

INTRODUCTION: Trichinellosis is still an epidemiological problem in Poland as well as in other countries in the European Union (EU). Across the EU, reporting cases oftrichinellosis is mandatory. In Poland, tirchinellosis is an endemic disease, occurring mainly in territories where it is customary to eat raw meat products prepared from pigs and wild boars. AIM: The aim of this work is to evaluate the epidemiological situation of trichinellosis in Poland in the year 2010 in comparison to previous years. MATERIAL AND METHODS: Cases of trichinellosis infections were classified according to criteria contained in the definition approved by the European Committee on 28th April 2008 amending the decision 2002/253/EC, and was introduced in Poland in 2009. Case definitions used in are available at http://www.pzh. gov.pl/oldpage/epimeld/inne/Def_PL2_Rob1 h.pdf. An infection was classified and reported as Trichinella spp. if the genus of Trichinella that caused the infection was not specified using molecular examination. RESULTS: In 2010 the number of registered human trichinellosis cases was similar to the average number of cases from the last several years, and it did not exceed 55 (the exceptions were in 2004 and 2007, when larger outbreaks occurred). In 2010, 51 Trichinella infections were registered, yielding an infection rate of 0.13 per 100 000 inhabitants. The infections occurred in 5 voivodeships (table 1). Using the criteria from the definitions, 41 cases were classified as probable and 10 were confirmed cases. Trichinella infections diagnoses were based on the presence of clinical symptoms and an epidemiological link. Serological diagnostic tests confirming the presence of Trichinella antibodies was performed in approximately 20% of the cases. Four individual cases were reported, along with 4 outbreaks in which a total of 47 people were infected? The infections were generally mild. Twenty-two infected persons were hospitalised (43%). Just like in previous years, no trichinellosis-related deaths were reported. The outbreaks of trichinellosis in 2010 occurred in voivodships where they are reported almost every year. Groups of 10 people and above were affected, consisting of family and friends. The incidence rate in three outbreaks ranged from 24% to 37%. In the Pomorskie voivodship this number was much higher--above 82%. This could mean that the infected products contained a large number of Trichinella larvae or that not all of the infected people were reached by the sanitary services. Small children are very rarely infected. In 2010 in the Wielkopolskie voivodeship, two such cases were reported in children who were 4 and 6 years old. Epidemiological investigation revealed that in the reported outbreaks the infections were most likely caused by the consumption of wild boar meat products, especially raw sausages. This finding was confirmed by analysing meat samples using the artificial digestion method. The particular genus of Trichinella was not specified in any of the cases and therefore they were classified as caused by Trichinella spp. The reason why infected wild boar meat was permitted to be consumed in two of the outbreaks was due to faulty carcass examination results. These examinations were conducted using the trichinoscopy method, which is still allowed in Poland despite being less sensitive than the artificial digestion method. In the Podlaskie voivodship outbreak the carcasses were not examined, and in the Pomorskie voivodship outbreak it could not be determined whether any examinations had been conducted. CONCLUSIONS. The annual occurrence of trichinellosis outbreaks and infections points to insufficient control of consumable meat products and lack of public awareness about the dangers of consuming raw/rare wild boar meat and pork that has not undergone prior veterinary inspection.

Epidemiology, diagnosis, treatment, and control of trichinellosis.

SUMMARY: Throughout much of the world, Trichinella spp. are found to be the causative agents of human trichinellosis, a disease that not only is a public health hazard by affecting human patients but also represents an economic problem in porcine animal production and food safety. Due to the predominantly zoonotic importance of infection, the main efforts in many countries have focused on the control of Trichinella or the elimination of Trichinella from the food chain. The most important source of human infection worldwide is the domestic pig, but, e.g., in Europe, meats of horses and wild boars have played a significant role during outbreaks within the past 3 decades. Infection of humans occurs with the ingestion of Trichinella larvae that are encysted in muscle tissue of domestic or wild animal meat. Early clinical diagnosis of trichinellosis is rather difficult because pathognomonic signs or symptoms are lacking. Subsequent chronic forms of the disease are not easy to diagnose, irrespective of parameters including clinical findings, laboratory findings (nonspecific laboratory parameters such as eosinophilia, muscle enzymes, and serology), and epidemiological investigations. New regulations laying down rules for official controls for Trichinella in meat in order to improve food safety for consumers have recently been released in Europe. The evidence that the disease can be monitored and to some extent controlled with a rigorous reporting and testing system in place should be motivation to expand appropriate programs worldwide.

[Trichinellosis in Poland in 2009]. / Wlosnica w Polsce w 2009 roku.

In the year 2009, thirty-six cases of human trichinellosis were registered in Poland, amounting to an incidence rate of 0,09 per 100 000 population. Of the registered cases 18 were confirmed and 18 were classed as probable according to EU definitions. There were no death cases resulting from the infections, however about 65,5% of the infected persons were hospitalized. Within the three registered outbreaks of trichinellosis two were caused by wild boar meat consumption and one by the consumption of meat products prepared from wild boar meat and pork. In all of the outbreaks, the contaminated meat products were prepared for household consumption. To avoid similar Trichinella outbreaks the polish public should be kept informed about the risks of unexamined pork and wild boar meat consumption. The importance of routine testing for trichinellosis in pig and wild boar carcasses should be adequately emphasized.

[Trichinellosis in Poland in 2008]. / Wlosnica w Polsce w 2008 roku.

In the year 2008 in Poland only 4 cases of human trichinellosis were registered. Although the course of the infection in all of the cases was benign the infected individuals were hospitalized. There were no outbreaks of trichinellosis. The incidence rate of the infection was 0.01 per 100 000 citizens.

Methods to assess the effect of meat processing on viability of Toxoplasma gondii: towards replacement of mouse bioassay by in vitro testing.

Consumption of meat containing viable tissue cysts is considered one of the main sources of human infection with Toxoplasma gondii. In contrast to fresh meat, raw meat products usually undergo processing, including salting and mixing with other additives such as sodium acetate and sodium lactate, which affects the viability of T. gondii. However, the experiments described in the literature are not always performed in line with the current processing methods applied in industry. It was our goal to study the effect of salting and additives according to the recipes used by industrial producers. Mouse or cat bioassay is the 'gold standard' to demonstrate the presence of viable T. gondii. However, it is costly, time consuming and for ethical reasons not preferred for large-scale studies.Therefore, we first aimed to develop an alternative for mouse bioassay that can be used to determine the effect of processing on the viability of T. gondii tissue cysts. The assays studied were (i) a cell culture method to determine the parasite's ability to multiply, and (ii) a propidium monoazide (PMA) dye-based assay to selectively detect DNA from intact parasites. Processing experiments were performed with minced meat incubated for 20 h with low concentrations of NaCl, sodium lactate and sodium acetate. NaCl appeared to be the most effective ingredient with only one or two out of eight mice infected after inoculation with pepsin-digest of portions processed with 1.0, 1.2 and 1.6% NaCl. Results of preliminary experiments with the PMA-based method were inconsistent and did not sufficiently discriminate between live and dead parasites. In contrast, the cell culture method showed promising results, but further optimization is needed before it can replace or reduce the number of mouse bioassays needed. In future, standardised in vitro methods are necessary to allow more extensive testing of product-specific processing methods, thereby providing a better indication of the risk of T. gondii infection for consumers.